Lactobacillus plantarum (LPsca12) enhances growth phenotype and muscle nutrition in abalone (Haliotis discus hannai) by modulating microbial function and metabolism through the Amino Acid-Driven Gut-Muscle Axis

Lactobacillus plantarum (LPsca12) enhances growth phenotype and muscle nutrition in abalone (Haliotis discus hannai) by modulating microbial function and metabolism through the Amino Acid-Driven Gut-Muscle Axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lactobacillus plantarum (LPsca12) enhances growth phenotype and muscle nutrition in abalone (Haliotis discus hannai) by modulating microbial function and metabolism through the Amino Acid-Driven Gut-Muscle Axis Yangyang He&, Lu Huang&, Shiwei Han, Xiaoyu Yu, Chenlu Wu, Xianan Dai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7232881/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Animal Microbiome → Version 1 posted 9 You are reading this latest preprint version Abstract Background With the continuous expansion of abalone ( Haliotis discus hannai ) aquaculture, improving growth rate and muscle quality has become an increasingly important concern. Probiotic supplementation represents a promising strategy; however, the effects of probiotics on abalone remain poorly understood, particularly concerning their mechanisms of action on muscle physiology. This study employed a multi-omics investigation to explore the impact of the Lactobacillus plantarum (LPsca12) strain on hepatic metabolism and gut microbiota composition in abalone, and the potential associations between intestinal metabolites and short-chain fatty acids (SCFAs) in muscle. Results Our findings reveal that (I) probiotic supplementation induced notable shifts in gut microbiota composition, thereby enhancing abalone growth performance and reducing lipid accumulation by promoting hepatic lipid metabolism, (II) the reshaped microbial community strongly linked to elevated concentrations of key amino acids in abalone, including L-leucine, L-isoleucine, L-valine, and L-arginine, which are considered potential precursors for SCFAs production, and (III) the elevated levels of SCFAs, such as acetate and propionate, in muscle tissue may activate the mTORC1 signaling pathway, thereby promoting protein synthesis and improving the nutritional status of abalone muscle. Conclusions The results indicate that LPsca12 exerts beneficial effects on abalone by enhancing growth performance and improving lipid metabolism and promoting muscle nutritional status through an amino acid-driven gut–muscle axis. Probiotics Abalone Gut microbiota Amino acids Muscle nutrition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background The abalone breeding scale has been continuously expanding to meet demands for food consumption and economic development [ 1 ]. Improving growth performance, survival rate, and flesh quality has become a primary focus in the abalone farming industry [ 2 ]. Previous research evidenced that both endogenous factors (such as genetic background and behavior) and exogenous factors (such as diet and culture environment) directly impact the growth and muscle quality of abalone [ 3 , 4 ]. During aquaculture, abalones are frequently subjected to intestinal metabolic disorders and viral infections, which negatively impact their health and impose significant constraints on their growth performance [ 5 – 7 ]. To optimize aquaculture efficiency, probiotics, as a green additive, are widely utilized in aquatic feed [ 8 ]. Probiotics have garnered considerable interest for their outstanding performance in preventing and alleviating diseases [ 9 ]. Probiotics have been shown to confer benefits via four primary mechanisms: enhancement of barrier integrity, modulation of immune responses, regulation of metabolic activities, and suppression of pathogenic microbes [ 10 , 11 ]. In addition, probiotics can exert strong ecological and evolutionary pressures that reshape the composition of the native gut microbiota [ 12 , 13 ]. Additionally, probiotics promote growth in fish and crustaceans, regulate the immune system, pre-digest antinutritional factors in feed, provide energy to epithelial cells, modulate gut microbiota, and enhance disease resistance [ 14 – 17 ]. As one of the earliest officially recognized microbial feed additives, lactic acid bacteria (LAB) has been applied in feed formulations over the past decades owing to its positive impacts on immunity, metabolism, and livestock growth [ 18 – 21 ]. However, the underlying mechanism has not been fully revealed, particularly given the limited research on the application of LAB in abalone. [ 22 ]. Gut microbiota is a diverse and sophisticated microbial community. It exhibits the highest dynamic diversity within the intestines and continuously evolves in response to the host’s physiological state and environmental changes [ 23 ]. This system is essential to vital physiological functions, including nutrient digestion and immune modulation, and is thus closely linked to the nutritional quality of abalone [ 24 ]. As the central organ of lipid metabolism, the liver is responsible for converting dietary fatty acids into polyunsaturated fatty acids required by muscle tissue. The gut microbiota directly influences hepatic lipid synthesis and transport through its metabolic by-products [ 25 , 26 ]. Studies in other animals have shown that the administration of Lactobacillus can reshape the gut microbial community and enhance intestinal metabolism. These microbial metabolites are transported to muscle tissue through the portal vein, where they regulate metabolic pathways involved in muscle synthesis, promote the activity of enzymes involved in protein biosynthesis, ultimately improving the nutritional supply for muscle formation [ 27 – 30 ]. Therefore, the intestinal microbiota exerts a significant impact on host muscle anabolism through the regulatory function of the gut–muscle axis. However, a substantial knowledge gap remains regarding how the gut microbiota supports aquatic animals, particularly in the context of abalone aquaculture, where current research is still highly limited [ 31 ]. It is worth noting that the digestive gland of abalone, a mollusk, shares functional similarities with the liver of vertebrates, especially in terms of its roles in digestion and metabolism [ 32 ]. Thus, it is referred to as the “liver” in this study. Based on this, we hypothesized that probiotics may enhance muscle nutrition of abalone by reshaping the gut microbiota structure, driving the synthesis of amino acid metabolites, subsequently reprogramming SCFAs in the muscle. To test this hypothesis, a feeding trial was conducted on abalone to evaluate muscle nutrition and lipid metabolism, while 16S rRNA sequencing and metabolomic analysis were employed to characterize variance in intestinal microbiota composition and metabolites. We also conducted a correlation analysis between microbial metabolism and growth status, aiming to enhance the theoretical framework supporting abalone aquaculture. 2. Materials and methods 2.1 Experimental design and animal management In total, 600 three-month-old abalone were obtained from Hangzhou Meiji Aquatic Products Trading Company and divided into two groups at random, 300 in each group, and 50 in each replicate. The control group (Con) received a basal diet, whereas the probiotic group (Pro) was provided with the same diet supplemented with the probiotic strain Lactobacillus plantarum (LPsca12). The LPsca12 were sprayed onto the basal feed daily and immediately fed to the abalone. The basal diet primarily consisted of fish meal, soybean meal (Shandong Haifeng Biotechnology Company, China) and Sargassum fusiforme (Zhejiang GOC Biotechnology Company Limited, China) (Table S1 ). LPsca12 was isolated in our laboratory and has been deposited at the China Center for Type Culture Collection (China). According to the aquaculture standards for abalone, each abalone was provided with 4 g of feed per day. The farming environment was maintained at a salinity of 25–28, a natural pH, and a temperature of 20°C with natural lighting. Water was changed daily, and waste was cleaned regularly. The experiment spanned 67 days, comprising a 7-day adaptation phase and a 60-day experimental phase. The experiment procedures were approved and following the laboratory animal welfare and management guidelines of Zhejiang Gongshang University, China. Each group contained 6 replicates, each of which was housed in a separate basket, and a total of 12 baskets were kept in the same room. Throughout the 60-day experimental period, body weight (BW), weight gain rate (WGR), length gain rate (LGR), and survival rate (SR) were recorded. All abalones in each group were individually measured on the first day. 2.2 Sample collection and biochemical analysis Following the 60-day experiment, three abalones per group were randomly sampled. After a 12 ± 0.5 h fasting period, the abalone were anesthetized on ice for 15 minutes and then dissected. Tissue samples were washed with cold physiological saline and gently dried using filter paper before being transferred to a beaker. The tissues were then finely chopped in an ice-cold environment and homogenized using a tissue grinder. The gut and its contents were promptly placed into liquid nitrogen and preserved at -80°C for further research. Crude protein (CP) and ether extract (EE) content in abalone muscle was carried out following AOAC (2000) methodologies, employing methods 954.01 for CP and 920.39 for EE. 2.3 Measurement of liver lipid metabolism Oil Red O staining was carried out by Biossci Biotechnologies Company Limited (China). Liver tissues were routinely processed after fixation in 4% paraformaldehyde to prepare 5 µm sections for staining, which were subsequently examined under an optical microscope. Aipathwell software was employed to quantify and analyze the proportion of positively stained regions. Triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), and the activities of liver enzymes, including lipase (LPS), hepatic lipase (HL), and lipoprotein lipase (LPL) were evaluated using assay kits (Nanjing Jiancheng Bioengineering Institute, China). 2.4 Short-chain fatty acids content in muscle Metabolite extraction was performed based on the chemical characteristics of multi-targeted metabolites. Freeze-dried samples (0.3 mg) were mixed with 300 µL of acetonitrile–water solution (1:1, v/v, containing internal standard, IS). After 10 minutes of ultrasonic extraction in an ice-cold environment, the mixture was placed at − 20°C for 30 minutes. After centrifugation (13,000 rpm, 10 min, 4°C), 80µL of the upper liquid was placed into a vial. Liquid chromatography analysis was performed on a Nexera UHPLC LC-30A system (SHIMADZU), utilizing an ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm). The injection volume was set to 5 µL. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile (B). The gradient elution program was as follows: 0 min, 10% B; 1 min, 10% B; 2 min, 25% B; 6 min, 35% B; 6.5 min, 95% B; 7.8 min, 95% B; 7.81 min, 10% B; 8.5 min, 10% B. Mass spectrometric detection was carried out using an AB SCIEX Selex ION Triple Quad™ 5500 System with an electrospray ionization (ESI) source operating in both positive and negative ionization modes. 2.5 16S rRNA gene sequencing Genomic DNA from feces was extracted with the MagPure Soil DNA LQ Kit (Shanghai Magen Biotechnology Company Limited, China). NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Company, USA) and agarose gel electrophoresis were used to evaluate the concentration and purity of extracted DNA. Then, the DNA was stored at -20 ° C and used as a template for PCR amplification of bacterial 16S rRNA genes using barcode specific primers and Takara Ex Taq high fidelity polymerase. General primers 343F (5′-TACGGRAGGCAGCAG-3′) and 798R (5′-AGGATCTAATCCT-3′) were used to amplify the V3–V4 variable region of the 16S rRNA gene for bacterial diversity analysis. PCR products were detected by agarose gel electrophoresis and purified by AMPure XP magnetic beads. Then use the purified product as a template for the second PCR amplification. After the second purification, the obtained product was quantified using a Qubit fluorometer and the concentration was normalized before sequencing. Sequencing was performed on the Illumina NovaSeq 6000 platform, resulting in paired end reads of 250 bp. The sequencing process was completed by Shanghai Ouyi Biotechnology Company Limited. 2.6 Measurement of Gut Microbial Metabolism Approximately 30 mg of the abalone fecal specimens was extracted with 400 µL of methanol-water solution (4:1, v/v) contaning 4 µg/mL L-2-chlorophenylalanine (IS). The sample underwent mechanical disruption using a bead mill and ultrasonic treatment in an ice-cold environment. After centrifugation, 300 µL of the upper liquid was harvested, dried and reconstituted in methanol–water (1:4, v/v), filtered through a 0.22 µm membrane, and preserved at − 80°C. All extracts were pooled to generate quality control (QC) samples. Metabolomic profiling was conducted using an ACQUITY UPLC I-Class Plus system integrated with a Q Exactive Plus mass spectrometer equipped with a HESI source (Thermo Fisher Scientific, USA) by a commercial service provider (Shanghai Luming Biotechnology Company Limited, China). Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column (1.8 µm, 2.1 × 100 mm). the mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with a flow rate of 0.35 mL/min. The total running time of gradient elution optimization was 16 min. the column temperature was set at 45°C, and the autosampler was kept at 10°C. The injection volume was 4 µL. Mass spectrometry was performed in both positive and negative ion modes, scanning over a mass range of 100–1200 m/z. The resolution settings were 70,000 for MS1 and 17,500 for MS2, with collision energies set at 10, 20, and 40 eV. 2.7 Targeted Metabolomics of Gut Amino Acids Metabolite extraction was performed according to the chemical characteristics of multi-targeted compounds. Add 400 µL of ice-cold methanol-water (4:1, v/v, containing IS) to the freeze-dried sample (30mg), then place in 2 steel balls and grind with a grinder (60Hz, 2 min). The samples were then ultrasonically extracted for 10 min in an ice-water bath and kept at − 20°C for 30 min. Liquid chromatography was carried out using a Nexera UHPLC LC-30A (SHIMADZU). Analysis was conducted using an ACQUITY UPLC BEH Amide (100 mm×2.1 mm, 1.7 µm). The injection volume is 1 µL. Mobile phase A is 0.2% formic acid-aqueous solution containing 10 mM ammonium formate, and mobile phase B is 0.2% formic acid-90% acetonitrile-water containing 10 mM ammonium formate. The gradient conditions were 0.3 mL/min:0 min, 100%B; 2 minutes, 100% B; 3 minutes, 90% B; 15 minutes, 85% B; 18 min, 40% B; 19 minutes, 40% B; 19.01 min, 100% B; 21 minutes, 100% B. Mass spectrometric detection was performed in positive multiple reaction mode. 2.8 Statistical analyses SPSS 27.0 (Chicago, IL, USA) was used for one-way analysis of variance (ANOVA) to assess the significance of differences between groups, with p-value < 0.05 as the criterion for statistical significance. Alpha diversity (Chao1 and Shannon index) of gut microbiota in each group was calculated and compared using t-test. calculating the Bray-Curtis distance of gut microbiota in different samples, and then PCoA and Adonis tests were performed to compare the differences between groups. Meanwhile, PCoA and Adonis tests were applied to analyze the differences of microbial metabolites between groups, and differential metabolites were selected according to p -value < 0.05. Spearman correlation was applied in testing the correlation among intestinal microbiota, lipid metabolism markers, and muscle nutrition. In the end, partial least squares path modeling (PLS-PM) was employed to evaluate the underlying mechanism of LPsca12 affecting growth performance and muscle nutrition. 3. Results 3.1 Abalone growth performance The results showed that the body weight (BW), weight gain rate (WGR), body length gain rate (LGR), and survival rate (SR) of the probiotic group (Pro) were remarkably improved compared to the control group (Con) (p < 0.05, Fig. 1 ), indicating that supplementing LPsca12 has a beneficial effect on the growth of abalone. 3.2 Fatty acid content in muscle SCFAs are key mediators of muscle metabolic processes and nutritional regulation. SCFAs measurements demonstrated clear metabolic distinctions between the Pro and Con groups (Fig. 2 B). Levels of SCFAs-including propionic acid, acetic acid, and 2,3-dihydroxy-3-methylbutanoic acid (DHMB acid) were markedly elevated in the Pro group, whereas these metabolites remained at relatively lower levels in the Con group. Among them, propionic acid and acetic acid showed a statistically significant increase in the Pro group (t-test, p < 0.05, Fig. 2 A). Besides, the enhancement of protein and lipid content in muscle can be measured to assess its nutritional value. Relative to the Con group, the Pro group demonstrated a marked enhancement in protein and lipid contents. (Fig. S3) These findings indicate that LPsca12 supplementation effectively enhances the nutritional metabolic profile of muscle. 3.3 LPsca12 supplementation reduces liver fat accumulation and enhances lipid metabolism in abalone Dietary supplementation with LPsca12 significantly reduced liver fat content in abalone, including TG, T-CHO, and LDL-C (p < 0.05, Fig. 3 A). A markedly lower percentage of Oil Red O-stained areas was detected in the Pro group liver tissue compared to the Con group (t-test, p < 0.05, Fig. 3 C). In addition, the activities of lipid decomposition-related enzymes (including LPS, LPL, and HL) were significantly elevated in the Pro group (t-test, p < 0.05, Fig. 3 B). These findings suggest that dietary supplementation with LPsca12 can reduce hepatic fat accumulation in abalone, thereby effectively controlling the fatty liver onset in abalone. 3.3 Consumption of LPsca12 supplements increases gut microbiota abundance Analysis of α diversity revealed that the Chao1 and Goods_coverage indices of the intestinal microbiota were marginally elevated in the Pro group in contrast to the Con group (t-test, p = 0.05, Fig. 4 D). Furthermore, a remarkable increase in the Shannon and Simpson diversity indices was noted in the Pro group (t-test, p < 0.05, Fig. 4 D). Analysis of β diversity using PCA and PCoA demonstrated that the Pro group formed a well-defined cluster (adonis test, p < 0.05, Fig. 4AB). The microbial communities in the Con and Pro groups were clearly separated and showed no overlap (Fig. 4 B). Additionally, the Pro group displayed greater intergroup variation in intestinal microbiota relative to the Con group (t-test, p = 0.05, Fig. 4 C). The observations clearly demonstrated that probiotic intake markedly alters the composition and diversity of the abalone intestinal microbiota. In abalone gut, Firmicutes constituted the predominant phylum, with Proteobacteria , Bacteroidota , and Fusobacteriota being less abundant. Probiotic supplementation had a significant impact on all these predominant phyla. The Pro group showed a marked elevation in the relative abundance of Firmicutes and a notable decrease in Proteobacteria (t-test, p < 0.05, Fig. 4 E). At the genus level, Mycoplasma was predominant, with Polaribacter , Psychrilyobacter , Vibrio , and Marivita following in abundance. LPsca12 notably influenced several bacterial genera, leading to decreased abundances of Mycoplasma and Polaribacter , while increasing those of Psychrilyobacter , Vibrio , and Marivita (t-test, p < 0.05, Fig. 4 F). 3.4 Functional alterations in the intestinal microbiota According to KEGG annotation, the result showed that LPsca12 significantly affected the abundance of several metabolic pathways in the abalone intestine (t-test, p < 0.05, Fig. 5 A). LPsca12 enhanced amino acid metabolism (including alanine, aspartate, glutamate, and arginine) and lipid metabolism (fatty acids and sphingolipids). In addition, LPsca12 strengthened several energy metabolism pathways (citrate cycle and oxidative phosphorylation), the digestion and degradation of carbohydrates (mannose and glucose), membrane transport (ABC transporters and secretion system), and nucleotide metabolism (purine and pyrimidine). Furthermore, we used network analysis to investigate the potential correction between gut microbial composition and these functional pathways (Fig. 5 B), and we found that intestinal microbial functions were strongly related to microbiota. Functional pathways related to intestinal bacteria were increased in their abundance after probiotic supplementation, such as tyrosine and leucine metabolism (ko00333), fatty acid metabolism (ko01212), and ABC transporters (ko02020) (Fig. 5 A). 3.5 The lipid metabolism and amino acid metabolism regulated by LPsca12 are significantly highlighted, but only amino acids improve muscle nutrition. PCA results demonstrated distinct clustering of the Pro and Con groups, with complete separation and no overlap (Fig. 6 A). A total of 341 metabolites were significantly reduced and 1755 significantly elevated in the Pro group compared to the Con group (Fig. 6 B). Differential metabolite analysis revealed significant changes in multiple host metabolites (T-test, p < 0.05, Fig. 6 C). LPsca12 improved amino acid metabolism (e.g. hydroxyprolyl-proline, glycil-prolyl-leucine), which are closely related to collagen renewal, peptide absorption, and muscle remodeling. LPsca12 also enhanced lipid metabolism (e.g., Conicasterol B, PE), organic heterocyclic compounds (e.g., Adenosine dialdehyde, Furagin), and organic oxidative compounds (e.g., 6-Acetyl-D-glucose), which are mainly involved in regulating energy metabolism, cell signaling, and antioxidant stress processes. Relative to the Con group, the Pro group predominantly exhibited upregulation in metabolic pathways related to key metabolic activities, including the citric acid cycle (TCA cycle), sulfur metabolism, taurine and sub taurine metabolism, arginine biosynthesis, phenylalanine metabolism, cysteine and methionine metabolism, and tyrosine metabolism. Additionally, the Pro Group also upregulated endocytosis related to cellular processes, sulfur relay systems related to genetic information processing, and neuroactive ligand receptor interactions related to environmental information processing (Fig. 6 D). Based on the metabolomic analysis, targeted quantification of amino acids (AAs) in abalone intestinal chyme from the Pro and Con groups was conducted. Compositional analysis revealed that 78.57% of the identified metabolites were classified as carboxylic acids and derivatives (S2), a class of compounds that are critically involved in muscle nutrient metabolism. Further targeted quantification showed that the concentrations of L-isoleucine, L-arginine, L-leucine, and L-valine were markedly elevated in the Pro group (Fig. 6 E), suggesting that probiotic supplementation enhanced the intestinal microbiota-mediated synthesis of key AAs. These observations support ongoing efforts to clarify the functional significance of SCFAs produced from AAs in muscle regulation. 3.6 Mechanisms of LPsca12 to regulate abalone Correlation analysis was first performed among the gut microbiota, lipid metabolism indicators, amino acid metabolism, and muscle SCFAs composition (Fig. 7 A). The results demonstrated that gut flora was markedly associated with liver fat content. Specifically, the activities of hepatic lipid metabolism-related enzymes, including LPS, LPL, and HL, were significantly correlated with the gut microbiota, showing positive associations particularly with the Psychrilyobacter , Vibrio, Marivita, Sedimentitalea , and Ruegeria . Regarding amino acid metabolism, there were also notable correlations between the intestinal microbiota and levels of AAs in the intestine. For example, Psychrilyobacter, Vibrio , and Ruegeria were positively correlated with several AAs, including L-pipecolic acid, L-proline, glycyl-L-proline, L-valine, L-alanine, Taurine, and Nα-acetyl-L-arginine, while Mycoplasma showed negative correlations with these metabolites. With respect to muscle SCFAs, propionate and acetate showed positive correlations with Psychrilyobacter, Marivita, Sedimentitalea , and Ruegeria , while exhibiting negative correlations with Planktotalea and Ahrensia . Additionally, propionate and acetate were significantly positively correlated with lipid metabolism-related enzymes, particularly with LPS activity. These two SCFAs also showed positive associations with certain amino acid metabolites, especially L-pipecolic acid, glycyl-L-proline, and Nα-acetyl-L-arginine. To gain deeper insights into the underlying mechanisms through which LPsca12 influences abalone growth performance and muscle nutritional quality, the PLS-PM approach was employed (Fig. 7 B). Data analysis demonstrated that dietary supplementation with LPsca12 was positively associated with hepatic enzyme activity, thereby reducing lipid accumulation in the liver. Moreover, LPsca12 exerted a beneficial effect on intestinal microbiota diversity, which in turn promoted AAs metabolism and increased the levels of SCFAs in abalone muscle. Collectively, LPsca12 improved growth performance by directly enhancing hepatic enzyme activity and indirectly modulating gut microbial diversity. 4. Discussion Farmed abalone typically require 3–5 years to reach the market size of 80 mm. Therefore, growth performance is one of the most important factors in the abalone industry [ 33 ]. Over past years, probiotics have gained increasing popularity as they provide an alternative source of beneficial microorganisms for aquaculture, which typically relies on terrestrial microbes [ 34 ]. Our research investigates the impact of probiotics supplementation on abalone growth performance, muscle nutritional composition, gut microbiota structure, and lipid metabolism, aiming to lay a groundwork for the application of probiotics in abalone farming. Our study found that LPsca12 treatment conferred beneficial effects on growth-related traits in abalone. In comparison with the Con group, body weight, shell length, and survival rate of the Pro group were significantly improved, which is in line with findings from studies on sea cucumbers and oysters [ 35 , 36 ]. SCFAs, particularly acetate and propionate, play direct roles in muscle anabolism, these metabolites are primarily synthesized in the host intestine and subsequently transported via the portal circulation to peripheral tissues such as muscle, where they contribute to energy supply, inflammatory modulation, and muscle protein synthesis [ 37 – 39 ]. Acetate has been shown to activate muscle cell metabolism and promote myofiber formation through AMPK signaling, while Propionate induces mTORC1 pathway activation, which is a pivotal regulator of protein biosynthesis [ 40 , 41 ]. However, studies on the effect of probiotics on SCFAs accumulation in abalone muscle remain limited. In our study, LPsca12 significantly elevated the content of acetate and propionate within abalone muscle tissue. Based on these findings, dietary LPsca12 may serve as a promising nutritional strategy to enhance muscle quality by elevating muscle-derived SCFAs levels and activating anabolic signaling pathways. Fat accumulation in the liver is a prevalent metabolic disorder in fish aquaculture, significantly affecting their growth performance and immune capacity, and may also cause liver and pancreatic damage, as well as energy metabolism disorders [ 42 , 43 ]. During the progression of lipid metabolism disorders, excessive circulating free fatty acids in adipose tissue are stored in the liver, leading to an imbalance in lipid homeostasis [ 44 ]. Elevated hepatic levels of TG and T-CHO are generally considered to be key features of lipid metabolism disorders and the formation of fatty liver [ 45 ]. Our study found that the levels of TG, T-CHO, and LDL-C in the liver of the Pro group were markedly reduced, indicating that hepatic lipid deposition was reduced, and lipid metabolism disorders were effectively improved. LPL, a rate-limiting enzyme in the metabolism of exogenous lipids, is an essential hydrolase that catalyzes the breakdown of triglycerides, releasing fatty acids for uptake and utilization by tissues [ 46 ]. HL regulates the liver's clearance andremodeling of lipoproteins and participates in tholesterol metabolism [ 47 ]. LPS functions in fat metabolism and transport by catalyzing the hydrolysis of lipids into glycerol and fatty acids [ 48 ]. The Pro group exhibited significantly higher hepatic LPL, HL, and LPS activity levels compared with the Con group, which helped to accelerate the clearance of exogenous lipids and optimize the overall lipid metabolism. Adding LPsca12 may improve the metabolic utilization efficiency of fatty acids by upregulating the expression and activity of liver lipases, thereby alleviating the lipid burden in the liver and promoting the restoration of lipid homeostasis. As a major regulator, the gut microbiota influences intestinal immune function, nutrient uptake, and overall host health [ 24 ]. However, it has been reported that its composition and diversity are influenced by farming environments, including water salinity, pH, feed sources, and seasonality [ 49 – 51 ]. In our study, we applied metagenomics to investigate the gut bacterial communities in abalone simultaneously. The analysis revealed a statistically significant increase in alpha diversity in the Progroup relative to the Con group. Combined with β diversity analysis, we inferred that LPsca12 contributes to increased species richness as well as diversity of the abalone gut microbiota. Studies have shown that Firmicutes and Bacteroidetes constitute the predominant phyla present guts of healthy, contributing significantly to host health, immune function, and internal homeostasis [ 52 , 53 ]. Our findings indicated that LPsca12 administration markedly elevated the abundance of Firmicutes and Bacteroidetes in the abalone intestinal microbiota. Additionally, some members of Psychrilyobacter and Vibrio were associated with healthy individuals [ 54 , 55 ]. Psychrilyobacter has previously been reported as probiotics that can improve abalone survival and immunity [ 56 ]. Furthermore, functional annotation based on KEGG pathway analysis indicated that LPsca12 supplementation could modulate the host microbiota composition and facilitate the proliferation of beneficial microbes, potentially through suppression of pathogenic bacterial colonization within the gastrointestinal tract. These findings highlight that LPsca12 can positively regulate the abalone gut microbiota and promote host health. Metabolomics offers an integrated view of metabolic profiles in biological specimens and enables detailed insights into host responses to diverse environmental stimuli and physiological conditions [ 57 – 60 ]. In our study, a total of 1,755 metabolites showed increased levels, while 341 exhibited decreased levels in the Pro relative to the Con group, and a notable enrichment of amino acids was observed in the Pro group. Beyond serving as essential substrates for protein synthesis, amino acids are critically involved in energy metabolism, neurotransmitter function, intestinal barrier maintenance, and the modulation of inflammatory responses [ 61 , 62 ]. Furthermore, amino acid–derived metabolites (e.g., SCFAs, amines, phenolic compounds) function as key modulators of gut homeostasis and host–microbe interactions [ 63 ]. Previous evidence have indicated that intestinal microbiota can profoundly impact host metabolism and muscle protein synthesis through the regulation of amino acid metabolism [ 64 ]. However, the mechanisms by which probiotics regulate amino acid metabolism and their derivatives to influence muscle nutrition in abalone remain insufficiently understood. In our study, we identified a potential association between gut amino acid metabolism and SCFAs production; however, the underlying mechanisms and their effects on host physiology warrant further investigation. Finally, we investigated the potential contribution of LPsca12 to muscle-related biosynthetic pathways. After probiotic supplementation, the intestinal microbiota of abalone exhibited significant enrichment, indicating an improved gut microbial environment. Based on this observation, we hypothesize that LPsca12 intervention modulated the microbial community, leading to detectable metabolic alterations. Notably, key nutrient metabolites, including branched-chain amino acids (L-leucine, L-isoleucine, and L-valine) and L-arginine, were significantly elevated in abalone tissues following probiotic treatment. Microbial catabolism of amino acids is known to generate ammonia, organic acids, and gaseous compounds, among which SCFAs are the most plentiful final metabolites [ 65 ]. This suggests that enhanced amino acid metabolism may effectively promote SCFAs biosynthesis, which is consistent with our findings. Among these amino acids, L-leucine and L-arginine are well-recognized activators of mTORC1 and may activate the mTORC1 complex via the Rag GTPase signaling pathway, thereby facilitating protein synthesis and myocyte growth, ultimately contributing to improved muscle nutritional status [ 66 ].In parallel, the elevated levels of SCFAs such as acetate and propionate in muscle tissue may activate the GPR41/43–AMPK–SIRT1 signaling axis, thereby enhancing mTORC1 responsiveness and synergistically regulating muscle energy metabolism and protein synthesis [ 67 , 68 ]. Moreover, our study revealed that LPsca12 significantly improved hepatic lipid metabolic parameters, characterized by decreased TG, T-CHO, and LDL-C and increased LPS, LPL, and HL levels. These changes suggest enhanced lipid clearance capacity and reduce ectopic lipid deposition, which may contribute to a more favorable metabolic milieu for muscle protein anabolism. Collectively, these findings suggest that LPsca12 may modulate muscle nutrition and metabolism in abalone through a “microbiota–AAs–SCFAs–mTORC1” signaling axis, possibly in concert with improved hepatic lipid metabolism. Although the precise molecular mechanisms remain to be elucidated, our study provides novel microecological insights for the regulation of muscle quality in aquatic animals. 5. Conclusion This study utilized a multi-omics strategy to comprehensively uncover the actions of probiotics on abalone growth performance and muscle nutrition, along with their potential underlying mechanisms. Our findings demonstrate that LPsca12 effectively improves the growth performance, hepatic lipid metabolism, and gut metabolic functions of abalone. Probiotic supplementation significantly modified the gut microbial community in abalone, characterized by the selective enrichment or depletion of specific microbial taxa, thereby enhancing intestinal metabolic activity. The reduction in hepatic fat accumulation observed in this study may result from alterations in lipid-metabolizing enzyme activity as well as shifts in the gut microbiota. Moreover, LPsca12 exhibited the capacity to promote nutrient deposition in muscle tissues, supported by the increased levels of SCFAs in muscle. This effect may be mediated by probiotic-induced microbial amino acid metabolism, which promotes SCFAs production and subsequently activates the mTORC1 signaling pathway, thereby enhancing muscle protein synthesis. Meanwhile, the improvement in hepatic lipid metabolism provides a more favorable metabolic environment. Collectively, these results shed new light on the regulatory function of the gut-muscle axis in abalone nutritional metabolism and underscore the prospect of LPsca12 as a functional feed additive for abalone farming. Declarations Data availability The datasets of 16S rRNA gene sequencing generated and/or analyzed in the current study are available in the NCBI Sequence Read Archive (SRA) database under the BioProject ID“PRJNA1279153”(The review link:https://www.ncbi.nlm.nih.gov/sra/PRJNA1279153) and the raw data of omics sequencing in this study have been deposited in the Genome Sequence Archive (GSA) under the accession number “OMIX010680” (The review link: https://ngdc.cncb.ac.cn/omix/release/OMIX010680). Author’s contributions Dafeng Song, Yangyang He, and Lu Huang designed the experiments. Shiwei Han and Chenlu Wu conducted the experiments. Xianan Dai and Zhizhong Lv collected the samples and performed the analysis of samples. Yangyang He, Lu Huang, and Shiwei Han analyzed the data. Yangyang He and Lu Huang wrote the paper. Dafeng Song, Yangyang He, and Lu Huang revised the manuscript. All authors read and approved the final manuscript. Funding This research was supported by Intensive Processing of Aquatic Products; Wenzhou Dongtou District Science and Technology Plan Project Study (award number:N2024Y04). Ethics approval and consent to participate Animal care and experimental procedures were approved by the Animal Care Committee of Zhejiang Gongshang University (Hangzhou, China) and followed the university’s guidelines for animal research. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Acknowledgments We thank OE Biotechnology Company Limited (Shanghai, China) for providing metabolomics services for this analysis. References Yin ZH, Liu JY, Yu WC, Shen YW, Gan Y, Chen YX, et al. Seasonal variation in two culture systems and genome-wide association analysis of taurine content in the foot muscles of the Pacific abalone. Aquaculture 2025;604. Chen P, Yang MX, Tian SJ, Wu ZH, Lin YJ, Mai KS, et al. 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Supplementary Files SupplementaryMaterial.docx GraphicalAbstract.jpg Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Animal Microbiome → Version 1 posted Editorial decision: Revision requested 26 Nov, 2025 Reviews received at journal 25 Nov, 2025 Reviewers agreed at journal 13 Oct, 2025 Reviews received at journal 03 Oct, 2025 Reviewers agreed at journal 22 Sep, 2025 Reviewers invited by journal 21 Sep, 2025 Editor assigned by journal 26 Aug, 2025 Submission checks completed at journal 29 Jul, 2025 First submitted to journal 28 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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12:03:52","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":462016,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/e545757149d986d8275960ab.png"},{"id":92713603,"identity":"5ce0953a-8bc4-419c-aadc-1fce6c129659","added_by":"auto","created_at":"2025-10-03 11:47:52","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":522354,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/db568e67daac8f19ff5b11a5.png"},{"id":92713401,"identity":"ac32a612-baf0-4f7e-aba6-b7f4acc32aa2","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":268181,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineGraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/b2ae4aa0d2cbfdbfc6061c17.png"},{"id":92713403,"identity":"1ad03d3c-eec5-4068-a731-b565cf6df6b5","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147498,"visible":true,"origin":"","legend":"","description":"","filename":"9edbd560e5fd44a6a455a3749768cce81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/959e7a50029297df89c89803.xml"},{"id":92713406,"identity":"43c162c5-1ac8-4987-8527-acb3558350ca","added_by":"auto","created_at":"2025-10-03 11:39:53","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":158839,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/3057c88dd2bea1dc2fc9e289.html"},{"id":92713592,"identity":"86d6e29d-ebd1-4fd1-82dc-1c79b00ac7f3","added_by":"auto","created_at":"2025-10-03 11:47:52","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":208033,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth performance and survival comparison. A Body weight. B Length gain rate. C Weight gain rate. D Survival rate. *** indicates adjusted p-value \u0026lt; 0.01. Weight gain rate (%) = (W\u003csub\u003efinal\u003c/sub\u003e−W\u003csub\u003einitial\u003c/sub\u003e)/W\u003csub\u003einitial\u003c/sub\u003e*100\u003csub\u003e. \u003c/sub\u003eLength gain rate (%) = (L\u003csub\u003efinal\u003c/sub\u003e−L\u003csub\u003einitial\u003c/sub\u003e)/L\u003csub\u003einitial\u003c/sub\u003e*100\u003csub\u003e.\u003c/sub\u003e Survival rate (%) = (N\u003csub\u003efinal\u003c/sub\u003e−N\u003csub\u003einitial\u003c/sub\u003e)/N\u003csub\u003einitial\u003c/sub\u003e*100.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/b6fd2716ddba2c75f490d46a.jpg"},{"id":92713375,"identity":"a74f49eb-8ab6-4120-a0b5-68f414f7f920","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":839735,"visible":true,"origin":"","legend":"\u003cp\u003eCompositions analysis of SCFAs in the muscle. A SCFAs content in muscle. B Clustered heatmap of SCFAs profiles in muscle. ** indicates adjusted p-value \u0026lt;0.05. *** indicates adjusted p-value \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/b0ec74df46630ff5c867585e.jpg"},{"id":92713376,"identity":"dce67e75-0f81-4544-90dc-02333472570f","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":724144,"visible":true,"origin":"","legend":"\u003cp\u003eHepatic Lipid Metabolism Analysis. A Lipid metabolism index. B Enzyme activities in the liver. *** indicates adjusted p-value \u0026lt;0.01. C Staining results of liver sections.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/3b0d67adc26a2b39aefdcbc8.jpg"},{"id":92713378,"identity":"4fed9dce-7529-4fa5-b473-d6ba4cdb9d04","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":598075,"visible":true,"origin":"","legend":"\u003cp\u003eDifference in gut microbiota. A The PCoA and adonis test based on the Bray–Curtis distance. B The PCA. C The Bray–Curtis distance. D Alpha diversity metrics. E Heatmap of differential bacterial phylum. F Heatmap of differential bacterial phylum. * indicates adjusted p-value = 0.05. *** indicates adjusted p-value \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/56b4a05105564f607068d65d.jpg"},{"id":92713377,"identity":"20218fe2-ea4e-4b7a-98e3-2d9b4facb44d","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1173489,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of functional pathways in intestinal microbiota. A Heatmap of functional pathway differences. B Network of correlations among functional pathways and bacterial communities.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/147e46192e84788e81e422f4.jpg"},{"id":92713381,"identity":"7e28608e-f801-429c-80a1-3bcbb67cb146","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":947462,"visible":true,"origin":"","legend":"\u003cp\u003eComprehensive analysis of metabolite differences in muscle. A The PCA analysis. B Volcano plot of differential metabolites. C Classification of differential metabolites. D KEGG enrichment analysis. E The AAs with significantly different contents in the gut. ** indicates adjusted p-value \u0026lt;0.05. *** indicates adjusted p-value \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/ed74f937cd2c162c3ee93dba.jpg"},{"id":92714311,"identity":"6792e8e4-5532-4363-859f-ace1b718d2f0","added_by":"auto","created_at":"2025-10-03 11:55:52","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":703742,"visible":true,"origin":"","legend":"\u003cp\u003eA Correlations of gut microbiota and amino acid metabolism. B Correlations of lipid metabolism indicators and composition of SCFAs in muscles. C Correlations of gut microbiota and lipid metabolism indicators. D Correlations of gut microbiota and composition of SCFAs in muscles. E Correlations of amino acid metabolism and composition of SCFAs in muscles. F PLS-PM reflecting the impacts of LPsca12 on gut microbiota, amino acid metabolism, liver function, and muscle nutrition in abalone. Only significant paths (p \u0026lt;0.05) are displayed. Blue arrows indicate positive correlations; brown arrows indicate negative correlations.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/89befd94af21b260765900f1.jpg"},{"id":102234091,"identity":"12bb7f5d-a25c-4527-9f77-67dd5be1971c","added_by":"auto","created_at":"2026-02-09 16:05:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6167535,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/ce916d02-9eef-4688-a023-4a92eba57696.pdf"},{"id":92713385,"identity":"44f13887-c376-419a-804e-4ff82572b890","added_by":"auto","created_at":"2025-10-03 11:39:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":270010,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/d2126262b2a4dff7529d2295.docx"},{"id":92713595,"identity":"96960411-9349-40c4-9296-88df4fb62239","added_by":"auto","created_at":"2025-10-03 11:47:52","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1688252,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7232881/v1/07fc713f2a80f2f61d98e139.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lactobacillus plantarum (LPsca12) enhances growth phenotype and muscle nutrition in abalone (Haliotis discus hannai) by modulating microbial function and metabolism through the Amino Acid-Driven Gut-Muscle Axis","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThe abalone breeding scale has been continuously expanding to meet demands for food consumption and economic development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Improving growth performance, survival rate, and flesh quality has become a primary focus in the abalone farming industry [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous research evidenced that both endogenous factors (such as genetic background and behavior) and exogenous factors (such as diet and culture environment) directly impact the growth and muscle quality of abalone [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. During aquaculture, abalones are frequently subjected to intestinal metabolic disorders and viral infections, which negatively impact their health and impose significant constraints on their growth performance [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo optimize aquaculture efficiency, probiotics, as a green additive, are widely utilized in aquatic feed [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Probiotics have garnered considerable interest for their outstanding performance in preventing and alleviating diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Probiotics have been shown to confer benefits via four primary mechanisms: enhancement of barrier integrity, modulation of immune responses, regulation of metabolic activities, and suppression of pathogenic microbes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, probiotics can exert strong ecological and evolutionary pressures that reshape the composition of the native gut microbiota [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, probiotics promote growth in fish and crustaceans, regulate the immune system, pre-digest antinutritional factors in feed, provide energy to epithelial cells, modulate gut microbiota, and enhance disease resistance [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As one of the earliest officially recognized microbial feed additives, lactic acid bacteria (LAB) has been applied in feed formulations over the past decades owing to its positive impacts on immunity, metabolism, and livestock growth [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the underlying mechanism has not been fully revealed, particularly given the limited research on the application of LAB in abalone. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGut microbiota is a diverse and sophisticated microbial community. It exhibits the highest dynamic diversity within the intestines and continuously evolves in response to the host\u0026rsquo;s physiological state and environmental changes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This system is essential to vital physiological functions, including nutrient digestion and immune modulation, and is thus closely linked to the nutritional quality of abalone [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. As the central organ of lipid metabolism, the liver is responsible for converting dietary fatty acids into polyunsaturated fatty acids required by muscle tissue. The gut microbiota directly influences hepatic lipid synthesis and transport through its metabolic by-products [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Studies in other animals have shown that the administration of \u003cem\u003eLactobacillus\u003c/em\u003e can reshape the gut microbial community and enhance intestinal metabolism. These microbial metabolites are transported to muscle tissue through the portal vein, where they regulate metabolic pathways involved in muscle synthesis, promote the activity of enzymes involved in protein biosynthesis, ultimately improving the nutritional supply for muscle formation [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, the intestinal microbiota exerts a significant impact on host muscle anabolism through the regulatory function of the gut\u0026ndash;muscle axis. However, a substantial knowledge gap remains regarding how the gut microbiota supports aquatic animals, particularly in the context of abalone aquaculture, where current research is still highly limited [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It is worth noting that the digestive gland of abalone, a mollusk, shares functional similarities with the liver of vertebrates, especially in terms of its roles in digestion and metabolism [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, it is referred to as the \u0026ldquo;liver\u0026rdquo; in this study.\u003c/p\u003e\u003cp\u003eBased on this, we hypothesized that probiotics may enhance muscle nutrition of abalone by reshaping the gut microbiota structure, driving the synthesis of amino acid metabolites, subsequently reprogramming SCFAs in the muscle. To test this hypothesis, a feeding trial was conducted on abalone to evaluate muscle nutrition and lipid metabolism, while 16S rRNA sequencing and metabolomic analysis were employed to characterize variance in intestinal microbiota composition and metabolites. We also conducted a correlation analysis between microbial metabolism and growth status, aiming to enhance the theoretical framework supporting abalone aquaculture.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental design and animal management\u003c/h2\u003e\u003cp\u003eIn total, 600 three-month-old abalone were obtained from Hangzhou Meiji Aquatic Products Trading Company and divided into two groups at random, 300 in each group, and 50 in each replicate. The control group (Con) received a basal diet, whereas the probiotic group (Pro) was provided with the same diet supplemented with the probiotic strain \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (LPsca12). The LPsca12 were sprayed onto the basal feed daily and immediately fed to the abalone. The basal diet primarily consisted of fish meal, soybean meal (Shandong Haifeng Biotechnology Company, China) and \u003cem\u003eSargassum fusiforme\u003c/em\u003e (Zhejiang GOC Biotechnology Company Limited, China) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). LPsca12 was isolated in our laboratory and has been deposited at the China Center for Type Culture Collection (China).\u003c/p\u003e\u003cp\u003eAccording to the aquaculture standards for abalone, each abalone was provided with 4 g of feed per day. The farming environment was maintained at a salinity of 25\u0026ndash;28, a natural pH, and a temperature of 20\u0026deg;C with natural lighting. Water was changed daily, and waste was cleaned regularly. The experiment spanned 67 days, comprising a 7-day adaptation phase and a 60-day experimental phase.\u003c/p\u003e\u003cp\u003e The experiment procedures were approved and following the laboratory animal welfare and management guidelines of Zhejiang Gongshang University, China. Each group contained 6 replicates, each of which was housed in a separate basket, and a total of 12 baskets were kept in the same room. Throughout the 60-day experimental period, body weight (BW), weight gain rate (WGR), length gain rate (LGR), and survival rate (SR) were recorded. All abalones in each group were individually measured on the first day.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Sample collection and biochemical analysis\u003c/h2\u003e\u003cp\u003eFollowing the 60-day experiment, three abalones per group were randomly sampled. After a 12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 h fasting period, the abalone were anesthetized on ice for 15 minutes and then dissected. Tissue samples were washed with cold physiological saline and gently dried using filter paper before being transferred to a beaker. The tissues were then finely chopped in an ice-cold environment and homogenized using a tissue grinder. The gut and its contents were promptly placed into liquid nitrogen and preserved at -80\u0026deg;C for further research. Crude protein (CP) and ether extract (EE) content in abalone muscle was carried out following AOAC (2000) methodologies, employing methods 954.01 for CP and 920.39 for EE.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Measurement of liver lipid metabolism\u003c/h2\u003e\u003cp\u003eOil Red O staining was carried out by Biossci Biotechnologies Company Limited (China). Liver tissues were routinely processed after fixation in 4% paraformaldehyde to prepare 5 \u0026micro;m sections for staining, which were subsequently examined under an optical microscope. Aipathwell software was employed to quantify and analyze the proportion of positively stained regions. Triglyceride (TG), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), and the activities of liver enzymes, including lipase (LPS), hepatic lipase (HL), and lipoprotein lipase (LPL) were evaluated using assay kits (Nanjing Jiancheng Bioengineering Institute, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Short-chain fatty acids content in muscle\u003c/h2\u003e\u003cp\u003eMetabolite extraction was performed based on the chemical characteristics of multi-targeted metabolites. Freeze-dried samples (0.3 mg) were mixed with 300 \u0026micro;L of acetonitrile\u0026ndash;water solution (1:1, v/v, containing internal standard, IS). After 10 minutes of ultrasonic extraction in an ice-cold environment, the mixture was placed at \u0026minus;\u0026thinsp;20\u0026deg;C for 30 minutes. After centrifugation (13,000 rpm, 10 min, 4\u0026deg;C), 80\u0026micro;L of the upper liquid was placed into a vial. Liquid chromatography analysis was performed on a Nexera UHPLC LC-30A system (SHIMADZU), utilizing an ACQUITY UPLC BEH C18 column (100 \u0026times; 2.1 mm, 1.7 \u0026micro;m). The injection volume was set to 5 \u0026micro;L. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile (B). The gradient elution program was as follows: 0 min, 10% B; 1 min, 10% B; 2 min, 25% B; 6 min, 35% B; 6.5 min, 95% B; 7.8 min, 95% B; 7.81 min, 10% B; 8.5 min, 10% B. Mass spectrometric detection was carried out using an AB SCIEX Selex ION Triple Quad\u0026trade; 5500 System with an electrospray ionization (ESI) source operating in both positive and negative ionization modes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 16S rRNA gene sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA from feces was extracted with the MagPure Soil DNA LQ Kit (Shanghai Magen Biotechnology Company Limited, China). NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Company, USA) and agarose gel electrophoresis were used to evaluate the concentration and purity of extracted DNA. Then, the DNA was stored at -20 \u0026deg; C and used as a template for PCR amplification of bacterial 16S rRNA genes using barcode specific primers and Takara Ex Taq high fidelity polymerase. General primers 343F (5\u0026prime;-TACGGRAGGCAGCAG-3\u0026prime;) and 798R (5\u0026prime;-AGGATCTAATCCT-3\u0026prime;) were used to amplify the V3\u0026ndash;V4 variable region of the 16S rRNA gene for bacterial diversity analysis. PCR products were detected by agarose gel electrophoresis and purified by AMPure XP magnetic beads. Then use the purified product as a template for the second PCR amplification. After the second purification, the obtained product was quantified using a Qubit fluorometer and the concentration was normalized before sequencing. Sequencing was performed on the Illumina NovaSeq 6000 platform, resulting in paired end reads of 250 bp. The sequencing process was completed by Shanghai Ouyi Biotechnology Company Limited.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Measurement of Gut Microbial Metabolism\u003c/h2\u003e\u003cp\u003eApproximately 30 mg of the abalone fecal specimens was extracted with 400 \u0026micro;L of methanol-water solution (4:1, v/v) contaning 4 \u0026micro;g/mL L-2-chlorophenylalanine (IS). The sample underwent mechanical disruption using a bead mill and ultrasonic treatment in an ice-cold environment. After centrifugation, 300 \u0026micro;L of the upper liquid was harvested, dried and reconstituted in methanol\u0026ndash;water (1:4, v/v), filtered through a 0.22 \u0026micro;m membrane, and preserved at \u0026minus;\u0026thinsp;80\u0026deg;C. All extracts were pooled to generate quality control (QC) samples. Metabolomic profiling was conducted using an ACQUITY UPLC I-Class Plus system integrated with a Q Exactive Plus mass spectrometer equipped with a HESI source (Thermo Fisher Scientific, USA) by a commercial service provider (Shanghai Luming Biotechnology Company Limited, China). Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column (1.8 \u0026micro;m, 2.1 \u0026times; 100 mm). the mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with a flow rate of 0.35 mL/min. The total running time of gradient elution optimization was 16 min. the column temperature was set at 45\u0026deg;C, and the autosampler was kept at 10\u0026deg;C. The injection volume was 4 \u0026micro;L. Mass spectrometry was performed in both positive and negative ion modes, scanning over a mass range of 100\u0026ndash;1200 m/z. The resolution settings were 70,000 for MS1 and 17,500 for MS2, with collision energies set at 10, 20, and 40 eV.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Targeted Metabolomics of Gut Amino Acids\u003c/h2\u003e\u003cp\u003eMetabolite extraction was performed according to the chemical characteristics of multi-targeted compounds. Add 400 \u0026micro;L of ice-cold methanol-water (4:1, v/v, containing IS) to the freeze-dried sample (30mg), then place in 2 steel balls and grind with a grinder (60Hz, 2 min). The samples were then ultrasonically extracted for 10 min in an ice-water bath and kept at \u0026minus;\u0026thinsp;20\u0026deg;C for 30 min. Liquid chromatography was carried out using a Nexera UHPLC LC-30A (SHIMADZU). Analysis was conducted using an ACQUITY UPLC BEH Amide (100 mm\u0026times;2.1 mm, 1.7 \u0026micro;m). The injection volume is 1 \u0026micro;L. Mobile phase A is 0.2% formic acid-aqueous solution containing 10 mM ammonium formate, and mobile phase B is 0.2% formic acid-90% acetonitrile-water containing 10 mM ammonium formate. The gradient conditions were 0.3 mL/min:0 min, 100%B; 2 minutes, 100% B; 3 minutes, 90% B; 15 minutes, 85% B; 18 min, 40% B; 19 minutes, 40% B; 19.01 min, 100% B; 21 minutes, 100% B. Mass spectrometric detection was performed in positive multiple reaction mode.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analyses\u003c/h2\u003e\u003cp\u003eSPSS 27.0 (Chicago, IL, USA) was used for one-way analysis of variance (ANOVA) to assess the significance of differences between groups, with p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as the criterion for statistical significance. Alpha diversity (Chao1 and Shannon index) of gut microbiota in each group was calculated and compared using t-test. calculating the Bray-Curtis distance of gut microbiota in different samples, and then PCoA and Adonis tests were performed to compare the differences between groups. Meanwhile, PCoA and Adonis tests were applied to analyze the differences of microbial metabolites between groups, and differential metabolites were selected according to p -value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Spearman correlation was applied in testing the correlation among intestinal microbiota, lipid metabolism markers, and muscle nutrition. In the end, partial least squares path modeling (PLS-PM) was employed to evaluate the underlying mechanism of LPsca12 affecting growth performance and muscle nutrition.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Abalone growth performance\u003c/h2\u003e\u003cp\u003eThe results showed that the body weight (BW), weight gain rate (WGR), body length gain rate (LGR), and survival rate (SR) of the probiotic group (Pro) were remarkably improved compared to the control group (Con) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that supplementing LPsca12 has a beneficial effect on the growth of abalone.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Fatty acid content in muscle\u003c/h2\u003e\u003cp\u003eSCFAs are key mediators of muscle metabolic processes and nutritional regulation. SCFAs measurements demonstrated clear metabolic distinctions between the Pro and Con groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Levels of SCFAs-including propionic acid, acetic acid, and 2,3-dihydroxy-3-methylbutanoic acid (DHMB acid) were markedly elevated in the Pro group, whereas these metabolites remained at relatively lower levels in the Con group. Among them, propionic acid and acetic acid showed a statistically significant increase in the Pro group (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Besides, the enhancement of protein and lipid content in muscle can be measured to assess its nutritional value. Relative to the Con group, the Pro group demonstrated a marked enhancement in protein and lipid contents. (Fig. S3) These findings indicate that LPsca12 supplementation effectively enhances the nutritional metabolic profile of muscle.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 LPsca12 supplementation reduces liver fat accumulation and enhances lipid metabolism in abalone\u003c/h2\u003e\u003cp\u003eDietary supplementation with LPsca12 significantly reduced liver fat content in abalone, including TG, T-CHO, and LDL-C (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A markedly lower percentage of Oil Red O-stained areas was detected in the Pro group liver tissue compared to the Con group (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, the activities of lipid decomposition-related enzymes (including LPS, LPL, and HL) were significantly elevated in the Pro group (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings suggest that dietary supplementation with LPsca12 can reduce hepatic fat accumulation in abalone, thereby effectively controlling the fatty liver onset in abalone.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Consumption of LPsca12 supplements increases gut microbiota abundance\u003c/h2\u003e\u003cp\u003eAnalysis of α diversity revealed that the Chao1 and Goods_coverage indices of the intestinal microbiota were marginally elevated in the Pro group in contrast to the Con group (t-test, p\u0026thinsp;=\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Furthermore, a remarkable increase in the Shannon and Simpson diversity indices was noted in the Pro group (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Analysis of β diversity using PCA and PCoA demonstrated that the Pro group formed a well-defined cluster (adonis test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;4AB). The microbial communities in the Con and Pro groups were clearly separated and showed no overlap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, the Pro group displayed greater intergroup variation in intestinal microbiota relative to the Con group (t-test, p\u0026thinsp;=\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The observations clearly demonstrated that probiotic intake markedly alters the composition and diversity of the abalone intestinal microbiota. In abalone gut, \u003cem\u003eFirmicutes\u003c/em\u003e constituted the predominant phylum, with \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e, and \u003cem\u003eFusobacteriota\u003c/em\u003e being less abundant. Probiotic supplementation had a significant impact on all these predominant phyla. The Pro group showed a marked elevation in the relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e and a notable decrease in \u003cem\u003eProteobacteria\u003c/em\u003e (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). At the genus level, \u003cem\u003eMycoplasma\u003c/em\u003e was predominant, with \u003cem\u003ePolaribacter\u003c/em\u003e, \u003cem\u003ePsychrilyobacter\u003c/em\u003e, \u003cem\u003eVibrio\u003c/em\u003e, and \u003cem\u003eMarivita\u003c/em\u003e following in abundance. LPsca12 notably influenced several bacterial genera, leading to decreased abundances of \u003cem\u003eMycoplasma\u003c/em\u003e and \u003cem\u003ePolaribacter\u003c/em\u003e, while increasing those of \u003cem\u003ePsychrilyobacter\u003c/em\u003e, \u003cem\u003eVibrio\u003c/em\u003e, and \u003cem\u003eMarivita\u003c/em\u003e (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Functional alterations in the intestinal microbiota\u003c/h2\u003e\u003cp\u003eAccording to KEGG annotation, the result showed that LPsca12 significantly affected the abundance of several metabolic pathways in the abalone intestine (t-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). LPsca12 enhanced amino acid metabolism (including alanine, aspartate, glutamate, and arginine) and lipid metabolism (fatty acids and sphingolipids). In addition, LPsca12 strengthened several energy metabolism pathways (citrate cycle and oxidative phosphorylation), the digestion and degradation of carbohydrates (mannose and glucose), membrane transport (ABC transporters and secretion system), and nucleotide metabolism (purine and pyrimidine). Furthermore, we used network analysis to investigate the potential correction between gut microbial composition and these functional pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and we found that intestinal microbial functions were strongly related to microbiota. Functional pathways related to intestinal bacteria were increased in their abundance after probiotic supplementation, such as tyrosine and leucine metabolism (ko00333), fatty acid metabolism (ko01212), and ABC transporters (ko02020) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.5 The lipid metabolism and amino acid metabolism regulated by LPsca12 are significantly highlighted, but only amino acids improve muscle nutrition.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePCA results demonstrated distinct clustering of the Pro and Con groups, with complete separation and no overlap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). A total of 341 metabolites were significantly reduced and 1755 significantly elevated in the Pro group compared to the Con group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Differential metabolite analysis revealed significant changes in multiple host metabolites (T-test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). LPsca12 improved amino acid metabolism (e.g. hydroxyprolyl-proline, glycil-prolyl-leucine), which are closely related to collagen renewal, peptide absorption, and muscle remodeling. LPsca12 also enhanced lipid metabolism (e.g., Conicasterol B, PE), organic heterocyclic compounds (e.g., Adenosine dialdehyde, Furagin), and organic oxidative compounds (e.g., 6-Acetyl-D-glucose), which are mainly involved in regulating energy metabolism, cell signaling, and antioxidant stress processes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRelative to the Con group, the Pro group predominantly exhibited upregulation in metabolic pathways related to key metabolic activities, including the citric acid cycle (TCA cycle), sulfur metabolism, taurine and sub taurine metabolism, arginine biosynthesis, phenylalanine metabolism, cysteine and methionine metabolism, and tyrosine metabolism. Additionally, the Pro Group also upregulated endocytosis related to cellular processes, sulfur relay systems related to genetic information processing, and neuroactive ligand receptor interactions related to environmental information processing (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eBased on the metabolomic analysis, targeted quantification of amino acids (AAs) in abalone intestinal chyme from the Pro and Con groups was conducted. Compositional analysis revealed that 78.57% of the identified metabolites were classified as carboxylic acids and derivatives (S2), a class of compounds that are critically involved in muscle nutrient metabolism. Further targeted quantification showed that the concentrations of L-isoleucine, L-arginine, L-leucine, and L-valine were markedly elevated in the Pro group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), suggesting that probiotic supplementation enhanced the intestinal microbiota-mediated synthesis of key AAs. These observations support ongoing efforts to clarify the functional significance of SCFAs produced from AAs in muscle regulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Mechanisms of LPsca12 to regulate abalone\u003c/h2\u003e\u003cp\u003eCorrelation analysis was first performed among the gut microbiota, lipid metabolism indicators, amino acid metabolism, and muscle SCFAs composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The results demonstrated that gut flora was markedly associated with liver fat content. Specifically, the activities of hepatic lipid metabolism-related enzymes, including LPS, LPL, and HL, were significantly correlated with the gut microbiota, showing positive associations particularly with the \u003cem\u003ePsychrilyobacter\u003c/em\u003e, \u003cem\u003eVibrio, Marivita, Sedimentitalea\u003c/em\u003e, and \u003cem\u003eRuegeria\u003c/em\u003e. Regarding amino acid metabolism, there were also notable correlations between the intestinal microbiota and levels of AAs in the intestine. For example, \u003cem\u003ePsychrilyobacter, Vibrio\u003c/em\u003e, and \u003cem\u003eRuegeria\u003c/em\u003e were positively correlated with several AAs, including L-pipecolic acid, L-proline, glycyl-L-proline, L-valine, L-alanine, Taurine, and Nα-acetyl-L-arginine, while \u003cem\u003eMycoplasma\u003c/em\u003e showed negative correlations with these metabolites. With respect to muscle SCFAs, propionate and acetate showed positive correlations with \u003cem\u003ePsychrilyobacter, Marivita, Sedimentitalea\u003c/em\u003e, and \u003cem\u003eRuegeria\u003c/em\u003e, while exhibiting negative correlations with \u003cem\u003ePlanktotalea\u003c/em\u003e and \u003cem\u003eAhrensia\u003c/em\u003e. Additionally, propionate and acetate were significantly positively correlated with lipid metabolism-related enzymes, particularly with LPS activity. These two SCFAs also showed positive associations with certain amino acid metabolites, especially L-pipecolic acid, glycyl-L-proline, and Nα-acetyl-L-arginine. To gain deeper insights into the underlying mechanisms through which LPsca12 influences abalone growth performance and muscle nutritional quality, the PLS-PM approach was employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Data analysis demonstrated that dietary supplementation with LPsca12 was positively associated with hepatic enzyme activity, thereby reducing lipid accumulation in the liver. Moreover, LPsca12 exerted a beneficial effect on intestinal microbiota diversity, which in turn promoted AAs metabolism and increased the levels of SCFAs in abalone muscle. Collectively, LPsca12 improved growth performance by directly enhancing hepatic enzyme activity and indirectly modulating gut microbial diversity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFarmed abalone typically require 3\u0026ndash;5 years to reach the market size of 80 mm. Therefore, growth performance is one of the most important factors in the abalone industry [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Over past years, probiotics have gained increasing popularity as they provide an alternative source of beneficial microorganisms for aquaculture, which typically relies on terrestrial microbes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our research investigates the impact of probiotics supplementation on abalone growth performance, muscle nutritional composition, gut microbiota structure, and lipid metabolism, aiming to lay a groundwork for the application of probiotics in abalone farming.\u003c/p\u003e\u003cp\u003eOur study found that LPsca12 treatment conferred beneficial effects on growth-related traits in abalone. In comparison with the Con group, body weight, shell length, and survival rate of the Pro group were significantly improved, which is in line with findings from studies on sea cucumbers and oysters [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSCFAs, particularly acetate and propionate, play direct roles in muscle anabolism, these metabolites are primarily synthesized in the host intestine and subsequently transported via the portal circulation to peripheral tissues such as muscle, where they contribute to energy supply, inflammatory modulation, and muscle protein synthesis [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Acetate has been shown to activate muscle cell metabolism and promote myofiber formation through AMPK signaling, while Propionate induces mTORC1 pathway activation, which is a pivotal regulator of protein biosynthesis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, studies on the effect of probiotics on SCFAs accumulation in abalone muscle remain limited. In our study, LPsca12 significantly elevated the content of acetate and propionate within abalone muscle tissue. Based on these findings, dietary LPsca12 may serve as a promising nutritional strategy to enhance muscle quality by elevating muscle-derived SCFAs levels and activating anabolic signaling pathways.\u003c/p\u003e\u003cp\u003eFat accumulation in the liver is a prevalent metabolic disorder in fish aquaculture, significantly affecting their growth performance and immune capacity, and may also cause liver and pancreatic damage, as well as energy metabolism disorders [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. During the progression of lipid metabolism disorders, excessive circulating free fatty acids in adipose tissue are stored in the liver, leading to an imbalance in lipid homeostasis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Elevated hepatic levels of TG and T-CHO are generally considered to be key features of lipid metabolism disorders and the formation of fatty liver [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our study found that the levels of TG, T-CHO, and LDL-C in the liver of the Pro group were markedly reduced, indicating that hepatic lipid deposition was reduced, and lipid metabolism disorders were effectively improved. LPL, a rate-limiting enzyme in the metabolism of exogenous lipids, is an essential hydrolase that catalyzes the breakdown of triglycerides, releasing fatty acids for uptake and utilization by tissues [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. HL regulates the liver's clearance andremodeling of lipoproteins and participates in tholesterol metabolism [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. LPS functions in fat metabolism and transport by catalyzing the hydrolysis of lipids into glycerol and fatty acids [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The Pro group exhibited significantly higher hepatic LPL, HL, and LPS activity levels compared with the Con group, which helped to accelerate the clearance of exogenous lipids and optimize the overall lipid metabolism. Adding LPsca12 may improve the metabolic utilization efficiency of fatty acids by upregulating the expression and activity of liver lipases, thereby alleviating the lipid burden in the liver and promoting the restoration of lipid homeostasis.\u003c/p\u003e\u003cp\u003eAs a major regulator, the gut microbiota influences intestinal immune function, nutrient uptake, and overall host health [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, it has been reported that its composition and diversity are influenced by farming environments, including water salinity, pH, feed sources, and seasonality [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In our study, we applied metagenomics to investigate the gut bacterial communities in abalone simultaneously. The analysis revealed a statistically significant increase in alpha diversity in the Progroup relative to the Con group. Combined with β diversity analysis, we inferred that LPsca12 contributes to increased species richness as well as diversity of the abalone gut microbiota. Studies have shown that \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e constitute the predominant phyla present guts of healthy, contributing significantly to host health, immune function, and internal homeostasis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Our findings indicated that LPsca12 administration markedly elevated the abundance of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e in the abalone intestinal microbiota. Additionally, some members of \u003cem\u003ePsychrilyobacter\u003c/em\u003e and \u003cem\u003eVibrio\u003c/em\u003e were associated with healthy individuals [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. \u003cem\u003ePsychrilyobacter\u003c/em\u003e has previously been reported as probiotics that can improve abalone survival and immunity [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Furthermore, functional annotation based on KEGG pathway analysis indicated that LPsca12 supplementation could modulate the host microbiota composition and facilitate the proliferation of beneficial microbes, potentially through suppression of pathogenic bacterial colonization within the gastrointestinal tract. These findings highlight that LPsca12 can positively regulate the abalone gut microbiota and promote host health.\u003c/p\u003e\u003cp\u003eMetabolomics offers an integrated view of metabolic profiles in biological specimens and enables detailed insights into host responses to diverse environmental stimuli and physiological conditions [\u003cspan additionalcitationids=\"CR58 CR59\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In our study, a total of 1,755 metabolites showed increased levels, while 341 exhibited decreased levels in the Pro relative to the Con group, and a notable enrichment of amino acids was observed in the Pro group. Beyond serving as essential substrates for protein synthesis, amino acids are critically involved in energy metabolism, neurotransmitter function, intestinal barrier maintenance, and the modulation of inflammatory responses [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Furthermore, amino acid\u0026ndash;derived metabolites (e.g., SCFAs, amines, phenolic compounds) function as key modulators of gut homeostasis and host\u0026ndash;microbe interactions [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Previous evidence have indicated that intestinal microbiota can profoundly impact host metabolism and muscle protein synthesis through the regulation of amino acid metabolism [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. However, the mechanisms by which probiotics regulate amino acid metabolism and their derivatives to influence muscle nutrition in abalone remain insufficiently understood. In our study, we identified a potential association between gut amino acid metabolism and SCFAs production; however, the underlying mechanisms and their effects on host physiology warrant further investigation.\u003c/p\u003e\u003cp\u003eFinally, we investigated the potential contribution of LPsca12 to muscle-related biosynthetic pathways. After probiotic supplementation, the intestinal microbiota of abalone exhibited significant enrichment, indicating an improved gut microbial environment. Based on this observation, we hypothesize that LPsca12 intervention modulated the microbial community, leading to detectable metabolic alterations. Notably, key nutrient metabolites, including branched-chain amino acids (L-leucine, L-isoleucine, and L-valine) and L-arginine, were significantly elevated in abalone tissues following probiotic treatment. Microbial catabolism of amino acids is known to generate ammonia, organic acids, and gaseous compounds, among which SCFAs are the most plentiful final metabolites [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. This suggests that enhanced amino acid metabolism may effectively promote SCFAs biosynthesis, which is consistent with our findings. Among these amino acids, L-leucine and L-arginine are well-recognized activators of mTORC1 and may activate the mTORC1 complex via the Rag GTPase signaling pathway, thereby facilitating protein synthesis and myocyte growth, ultimately contributing to improved muscle nutritional status [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].In parallel, the elevated levels of SCFAs such as acetate and propionate in muscle tissue may activate the GPR41/43\u0026ndash;AMPK\u0026ndash;SIRT1 signaling axis, thereby enhancing mTORC1 responsiveness and synergistically regulating muscle energy metabolism and protein synthesis [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Moreover, our study revealed that LPsca12 significantly improved hepatic lipid metabolic parameters, characterized by decreased TG, T-CHO, and LDL-C and increased LPS, LPL, and HL levels. These changes suggest enhanced lipid clearance capacity and reduce ectopic lipid deposition, which may contribute to a more favorable metabolic milieu for muscle protein anabolism.\u003c/p\u003e\u003cp\u003eCollectively, these findings suggest that LPsca12 may modulate muscle nutrition and metabolism in abalone through a \u0026ldquo;microbiota\u0026ndash;AAs\u0026ndash;SCFAs\u0026ndash;mTORC1\u0026rdquo; signaling axis, possibly in concert with improved hepatic lipid metabolism. Although the precise molecular mechanisms remain to be elucidated, our study provides novel microecological insights for the regulation of muscle quality in aquatic animals.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study utilized a multi-omics strategy to comprehensively uncover the actions of probiotics on abalone growth performance and muscle nutrition, along with their potential underlying mechanisms. Our findings demonstrate that LPsca12 effectively improves the growth performance, hepatic lipid metabolism, and gut metabolic functions of abalone. Probiotic supplementation significantly modified the gut microbial community in abalone, characterized by the selective enrichment or depletion of specific microbial taxa, thereby enhancing intestinal metabolic activity. The reduction in hepatic fat accumulation observed in this study may result from alterations in lipid-metabolizing enzyme activity as well as shifts in the gut microbiota. Moreover, LPsca12 exhibited the capacity to promote nutrient deposition in muscle tissues, supported by the increased levels of SCFAs in muscle. This effect may be mediated by probiotic-induced microbial amino acid metabolism, which promotes SCFAs production and subsequently activates the mTORC1 signaling pathway, thereby enhancing muscle protein synthesis. Meanwhile, the improvement in hepatic lipid metabolism provides a more favorable metabolic environment. Collectively, these results shed new light on the regulatory function of the gut-muscle axis in abalone nutritional metabolism and underscore the prospect of LPsca12 as a functional feed additive for abalone farming.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets of 16S rRNA gene sequencing generated and/or analyzed in the current study are available in the NCBI Sequence Read Archive (SRA) database under the BioProject ID\u0026ldquo;PRJNA1279153\u0026rdquo;(The review link:https://www.ncbi.nlm.nih.gov/sra/PRJNA1279153) and the raw data of omics sequencing in this study have been deposited in the Genome Sequence Archive (GSA) under the accession number \u0026ldquo;OMIX010680\u0026rdquo; (The review link: https://ngdc.cncb.ac.cn/omix/release/OMIX010680).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDafeng Song, Yangyang He, and Lu Huang designed the experiments. Shiwei Han and Chenlu Wu conducted the experiments. Xianan Dai and Zhizhong Lv collected the samples and performed the analysis of samples. Yangyang He, Lu Huang, and Shiwei Han analyzed the data. Yangyang He and Lu Huang wrote the paper. Dafeng Song, Yangyang He, and Lu Huang revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Intensive Processing of Aquatic Products; Wenzhou Dongtou District Science and Technology Plan Project Study (award number:N2024Y04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal care and experimental procedures were approved by the Animal Care Committee of Zhejiang Gongshang University (Hangzhou, China) and followed the university\u0026rsquo;s guidelines for animal research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank OE Biotechnology Company Limited (Shanghai, China) for providing metabolomics services for this analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYin ZH, Liu JY, Yu WC, Shen YW, Gan Y, Chen YX, et al. 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Ageing Res Rev 2024;98:102316.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"animal-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amic","sideBox":"Learn more about [Animal Microbiome](http://animalmicrobiome.biomedcentral.com)","snPcode":"42523","submissionUrl":"https://submission.nature.com/new-submission/42523/3","title":"Animal Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Probiotics, Abalone, Gut microbiota, Amino acids, Muscle nutrition","lastPublishedDoi":"10.21203/rs.3.rs-7232881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7232881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eWith the continuous expansion of abalone (\u003cem\u003eHaliotis discus hannai\u003c/em\u003e) aquaculture, improving growth rate and muscle quality has become an increasingly important concern. Probiotic supplementation represents a promising strategy; however, the effects of probiotics on abalone remain poorly understood, particularly concerning their mechanisms of action on muscle physiology. This study employed a multi-omics investigation to explore the impact of the \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (LPsca12) strain on hepatic metabolism and gut microbiota composition in abalone, and the potential associations between intestinal metabolites and short-chain fatty acids (SCFAs) in muscle.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOur findings reveal that (I) probiotic supplementation induced notable shifts in gut microbiota composition, thereby enhancing abalone growth performance and reducing lipid accumulation by promoting hepatic lipid metabolism, (II) the reshaped microbial community strongly linked to elevated concentrations of key amino acids in abalone, including L-leucine, L-isoleucine, L-valine, and L-arginine, which are considered potential precursors for SCFAs production, and (III) the elevated levels of SCFAs, such as acetate and propionate, in muscle tissue may activate the mTORC1 signaling pathway, thereby promoting protein synthesis and improving the nutritional status of abalone muscle.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe results indicate that LPsca12 exerts beneficial effects on abalone by enhancing growth performance and improving lipid metabolism and promoting muscle nutritional status through an amino acid-driven gut\u0026ndash;muscle axis.\u003c/p\u003e","manuscriptTitle":"Lactobacillus plantarum (LPsca12) enhances growth phenotype and muscle nutrition in abalone (Haliotis discus hannai) by modulating microbial function and metabolism through the Amino Acid-Driven Gut-Muscle Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 11:39:47","doi":"10.21203/rs.3.rs-7232881/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-27T02:56:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T06:34:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52188556596897966878648996248371320563","date":"2025-10-13T14:43:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T13:59:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247735994526276552543856320308164855091","date":"2025-09-22T11:03:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-22T00:21:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-26T16:18:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-29T06:20:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Animal Microbiome","date":"2025-07-28T10:33:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"animal-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amic","sideBox":"Learn more about [Animal Microbiome](http://animalmicrobiome.biomedcentral.com)","snPcode":"42523","submissionUrl":"https://submission.nature.com/new-submission/42523/3","title":"Animal Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0788c39d-d41e-40a7-86af-93b0659a7bc6","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:01:56+00:00","versionOfRecord":{"articleIdentity":"rs-7232881","link":"https://doi.org/10.1186/s42523-026-00521-4","journal":{"identity":"animal-microbiome","isVorOnly":false,"title":"Animal Microbiome"},"publishedOn":"2026-02-03 15:58:57","publishedOnDateReadable":"February 3rd, 2026"},"versionCreatedAt":"2025-10-03 11:39:47","video":"","vorDoi":"10.1186/s42523-026-00521-4","vorDoiUrl":"https://doi.org/10.1186/s42523-026-00521-4","workflowStages":[]},"version":"v1","identity":"rs-7232881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7232881","identity":"rs-7232881","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}